1. Field of the Invention
The present invention relates to a probe medium that includes a probe capable of specifically binding to a target substance and a method of producing the probe medium. In addition, the present invention relates to a probe medium useful for effectively immobilizing a probe on a substrate, a method of producing the probe medium, and a method of immobilizing a probe using the probe medium. Furthermore, the present invention relates to a probe-immobilized substrate obtained by immobilizing a probe on a substrate, and a detection element and a detection method for detecting a target substance using the probe-immobilized substrate.
2. Related Background Art
Recent advances in the field of biotechnology including genetic engineering and molecular biology have made possible diagnostics of infectious diseases, cancers, genetic disorders, and so on in DNA and RNA levels. As one of tools used for the diagnosis with nucleic acids such as DNA or RNA, attentions have been focused on DNA chips and DNA arrays. In the diagnostic tools including DNA chips and DNA arrays, probes such as nucleic acids are immobilized on a substrate and then the probes are hybridized with their target substances to detect those target substances. The probes capable of specifically binding to the target substances such as DNA, RNA, and nucleic acids are soluble in water and thus they can be easily dissolved in water, but hardly soluble in organic solvents such as ethyl alcohol, isopropyl alcohol, isoamyl alcohol, dipropylene glycol, and chloroform.
For the formation of a substrate on which a probe such as a DNA chip or a DNA array prepared in advance is immobilized by a spotting method or the like, conventionally a probe medium is prepared by dissolving the probe in an aqueous solution containing water or a mixture of water and a pH-adjusting material when the probe is immobilized on the substrate, and the probe medium is contacted with the substrate to immobilize the probe on the substrate. More specifically, for example, Japanese Patent Application Laid-Open No. H08-23975 describes a method of immobilizing a probe on a substrate, in which a probe aqueous solution is prepared by dissolving DNA in water and the probe medium thus prepared is dispensed and dropped into a surface-treated well-plate to immobilize the probe on the substrate. In addition, Japanese Patent Application Laid-Open No. H05-192198 describes a method of preparing a probe medium by adjusting the concentration of DNA by dissolving the DNA in a 10-mM Tris-HCl (pH 7.6)/1-mM EDTA solution, adding four-fold volumes of H2O and five-fold volumes of an immobilization buffer (1.5 M NaCl, 0.3 M Tris-HCl (pH 8.0), and 0.3 M MgCl2) to the DNA in buffer, and mixing them together. In Japanese Patent Application Laid-Open No. 2000-146971, furthermore, there is described a method in which an aqueous solution of oligonucleotide biotin-introduced to 5′-end is prepared, dotted on an isocyanated slide glass, and immobilized in an incubator at 37° C. for 15 minutes. Moreover, in Japanese Patent No. 2794728, a single-stranded DNA is serially diluted with a TE buffer (10 mM Tris-HCl (pH 7.5)/1 mM EDTA) to prepare a probe medium. The prepared probe medium was dotted on a nitrocellulose film, followed by air- and heat-drying to immobilize the DNA on a substrate.
However, among probe media including probes that are conventionally used and capable of specifically binding to the respective target substances, as a medium for immobilizing a probe on a substrate, there is no probe medium in which the probe is dissolved in an organic solvent where the probe is insoluble. Among those, furthermore, there is no probe medium containing a substance capable of solubilizing a probe in an organic solvent where the probe is insoluble. In each of the probe media conventionally used, a probe capable of specifically binding to a target substance is dissolved in water or an aqueous solution that contains water, a pH-adjusting substance, and an adsorption-lowering substance. Furthermore, it is also known that the addition of a small amount of a glycol-based solvent or an alcohol-based solvent is preferable to dot the probe medium on a substrate by spotting or the like. However, there is no probe medium prepared such that a probe such as nucleic acids of DNA or RNA is dissolved in a probe-insoluble organic solvent. Therefore, because of insolubility of the probe in an organic solvent, it has been difficult to realize a probe medium only including an organic solvent without containing water even though it is suitable to preferably dot the probe medium on the substrate.
Dissolving a probe such as nucleic acids of DNA or RNA in an organic solvent is known to extract the probe from a sample. However, as a probe medium to be used for immobilizing the probe on a substrate, no probe medium in which a probe is dissolved in an organic solvent has been known. In addition, for binding a probe such as nucleic acids of DNA or RNA to a carrier capable of binding to nucleic acids, the carrier suspended in a solution containing a surfactant is used to bind the nucleic acids for the extraction thereof. However, there is no case in which a probe is dissolved in a solution containing a surfactant and then the resultant solution is used as a probe medium.